1. Field of the Invention
The present invention relates to a reagent for measuring reticulocytes and also to a method of measuring them. More particularly, it relates to a reagent for measuring reticulocytes and a reticulocyte maturation index in blood in the field of clinical tests and also to a method of measuring them.
2. Description of the Related Arts
Reticulocytes are young erythrocytes immediately after a release of denucleated erythroblastic cells in bone marrow into peripheral blood. Characteristics of reticulocytes are that, as traces of maturing steps of erythrocytes, they retain, as residual substances in their cells, cell organelles such as ribosome and mitochondria and a small amount of RNA, which are not contained in matured erythrocytes.
In the field of clinical tests, it is very important to classify and count reticulocytes for grasping hematopoietic state in bone marrow of patients. In a normal and healthy person where hematopoiesis in bone marrow is normal, the number of reticulocytes occupies 0.5-3.0% of total numbers of erythrocytes. However, in the case where the hematopoiesis in bone marrow is abnormal, for example, where the hematopoiesis in bone marrow is inhibited, the number of reticulocytes decreases, while where the hematopoiesis in bone marrow is promoted, the number of reticulocytes increases. To be more specific, it decreases during the course of chemotherapy for a plastic anemia and malignant tumor while they increase in the case of hemolytic anemia and the like.
One of conventional methods for counting reticulocytes is a manual method in which a blood sample is mixed with a staining solution containing a basic dye such as New Methylene Blue (NMB) or Brilliant Cresyl Blue (BCB) to precipitate and to stain the above-mentioned residual substances contained in reticulocytes and each cell is observed under a microscope to discriminate reticulocytes from matured erythrocytes.
However, there are problems in such a manual method that an operation of preparing the sample is troublesome, individual difference for the discrimination of the cells takes place among medical technician and the statistic error is big due to the fact that the number of counted cells is small.
In order to solve these problems, a method (flow cytometry) has been conducted. In said method, reticulocytes are subjected to fluorescence staining using a fluorescent basic dye instead of the above-mentioned basic dye, the forward scattered light intensity and fluorescence intensity of cells are measured by a flow cytometer and matured erythrocytes and reticulocytes are discriminated, classified and counted mostly by means of the difference in the fluorescence intensities between them. As the dye used for this method, Acridine Orange, Thiazole Orange, Auramine O, etc. are well known.
In addition, in this method, it is also possible to classify and count reticulocytes depending upon the degree of maturation by means of the fluorescence intensity of reticulocytes whereby the maturation index of reticulocytes can be calculated. Since it has been known that the proportion of reticulocytes having a high fluorescence intensity or the proportion of youngest reticulocytes is useful as an index for the recovery of hematopoietic ability of bone marrow, utilization of said method has been expected.
However, an argon laser which is very expensive and is large in size is required as a light source for exciting the fluorescent dye and, therefore, there is a problem that the apparatus itself becomes expensive and large too. In addition, the above-mentioned dyes which can be used for the flow cytometry except Auramine 0 require a staining time of five minutes or more for staining reticulocytes. Especially, it has been reported by Lee, L. G. et al: Cytometry; 7:508-517(1986) that Thiazole Orange requires a staining time of 60 minutes or more and, therefore, it is difficult to fully automate the preparation of the sample whereby there is a problem in view of economy of manpower. Thus, the preparation of the sample is carried out by hands and, therefore, there is a problem that the data will vary depending upon technique of a person who prepares the sample and that, especially in the case of the maturation index of reticulocytes, large difference in the data may occur.
In the U. S. Pat. No. 5,360,739, there is a disclosure on a flow cytometric method in which reticulocytes are determined by measuring scatter light intensity and absorbance or fluorescence intensity using a He/Ne laser which is a relatively cheap light source and Oxazin 750 as a dye. However, the problem of abnormal lymphocytes which will be mentioned later is not described there.
In the meanwhile, the present inventor has also conducted an intensive study on reagents and methods for accurate and quick measurement of reticulocytes and reticulocyte maturation index by use of a flow cytometric method with a less expensive and small light source but there is still a problem that, especially in samples from patients where leukocytes are abnormal in number or type, for example, abnormal lymphocytes appear, the fluorescence intensity of lymphocytes is nearly the same as that of reticulocytes and, therefore, discrimination between them is difficult. Accordingly, neither reagent nor method for accurate and quick measurement has been established so far for the case where the discrimination between leukocytes and reticulocytes is difficult. Incidentally, this fact is already suggested in an instruction manual for a Retic-Count.TM. which is a kit for measuring of erythrocytes by a flow cytometry using an argon laser as a light source and is available in the market already. In this manual, it is mentioned that, when a sample containing an abnormal increase in leukocyte number is measured, confirmation by another method is necessary.